草莓EST-SSR标记开发及在品种遗传多样性分析中的应用

 【目的】探讨草莓EST序列中SSR位点的分布规律，开发草莓EST-SSR引物，并分析其在草莓品种遗传多样性研究中的应用。【方法】从NCBI公共数据库下载草莓表达序列标签（expressed sequence tag，EST）55 750条，利用MISA软件查找SSR位点，并利用Primer3.0 Plus软件设计60对引物，通过非变性聚丙烯酰胺凝胶（PAGE）研究这些SSR引物的PCR扩增特点，并对部分扩增产物进行克隆和测序，以验证其真实性。【结果】55 750条草莓EST序列含有SSR位点的序列1 334条，SSR位点1 490个。其中，二核苷酸、三核苷酸和六核苷酸重复是最主要的SSR类型，分别占36.17%、26.51%和19.87%。60对引物中有42对引物能在20个草莓品种中扩增出理想的PCR产物，其中36对引物扩增条带具有多态性。利用11对验证的EST-SSR引物分析了20个草莓品种的亲缘关系。【结论】草莓EST-SSR标记的开发对于草莓品种鉴定与遗传多样性分析具有重要应用价值。

Development of EST-Derived SSR Markers and Their Application in Strawberry Genetic Diversity Analysis

【Objective】 The objective of this study was to analyze the SSR distribution in strawberry ESTs and to develop new EST-derived SSR markers, and application of EST-SSR markers in strawberry genetic diversity analysis was also validated. 【Method】 A total of 55 750 EST sequences of strawberry were obtained from NCBI. These sequences were screened by using MISA software to search for SSR motifs. Sixty pairs of primers were designed by the software Primer3.0 Plus. The PCR products of these primers were detected by PAGE and some of them were recovered for sequencing. 【Result】 A total of 1 490 SSRs were identified from 1 334 strawberry EST sequences. The dinucleotide, trinucleotide and hexanucleotide repeats were the dominant types with the frequency of 36.17%, 26.51% and 19.87%, respectively. Among the 60 EST-SSR primers, 42 amplified distinct bands and expected products, and 36 were polymorphic. Eleven polymorphic EST-SSR amplicons were validated by resequencing the PCR products. A dendrogram tree generated by the 11 validated polymorphic EST-SSR markers revealed reasonable genetic relationships among 20 strawberry cultivars. 【Conclusion】 It is an efficient and economic way to develop SSR markers from the strawberry EST sequences, and the EST-SSR markers can be used in strawberry genetic analysis.